"Monkey wrench" molecule jams tuberculosis protein

Tuberculosis has now surpassed HIV worldwide as the leading cause of death due to infectious disease. The bacteria that causes this disease, Mycobacterium tuberculosis (Mtb), is quickly developing resistance to currently available antibiotics, increasing the urgency for discovery of new drugs.

Scientists at the U.S. Department of Energy’s (DOE) Argonne National Laboratory were part of a recent discovery of a new molecule called an inhibitor that attacks tuberculosis-causing bacteria by cutting off its production of a chemical necessary for its survival.

Creating antibiotics involves developing inhibitors that can effectively block essential bacterial processes. Equally important is to keep finding novel pathways to target so that when the bacteria eventually adapt to resist one antibiotic, there are still other avenues available to exploit.

“The inhibitor actually works in quite a sneaky way.”

The new study, a collaboration between Argonne, the University of Chicago and the Broad Institute of Harvard and MIT, identified an inhibitor that works by blocking the action of an important protein in Mtb. The inhibitor binds to a channel within the protein that connects its two parts, killing the bacteria through an ironic twist. The inhibitor forces one part of the protein to produce the components necessary to create an essential chemical, tryptophan, while at the same time reshaping the channel to intercept those ingredients before the other part of the protein can use them.

These types of inhibitors, called allosteric inhibitors, do not bind to the part of the protein actually responsible for producing the target chemical. Instead, they act as a monkey wrench jammed into other less obvious parts of the complicated machinery of the bacteria. The inhibitor identified in this research, described in a recent paper in Nature Chemical Biology, is now one of the most deeply studied allosteric inhibitors to date.

The targeted chemical, tryptophan, is a substance that is crucial in giving all living cells their structure and protecting them from their surroundings. Human cells have to rummage tryptophan from food or bacteria in the gut, but Mtb cells can manufacture their own tryptophan using a complex protein called tryptophan synthase. “We knew that one way of killing these bacteria was finding a way to block the tryptophan synthase,” said Karolina Michalska, an Argonne crystallographer and author of the study.

Before the collaboration began, a group of scientists at the Broad Institute put a small amount of the bacteria in petri dishes and placed different potential inhibitors in each one. Out of a library of over 80,000 tested potential inhibitors, only a few killed the Mtb. The scientists suspected that one of these inhibitors shut down the Mtb’s tryptophan synthase, but they needed to see what was happening on the molecular level.

Argonne scientists heard about the work at the Broad Institute, and the two groups began to collaborate. Argonne produced tryptophan synthase for the Broad Institute to use for testing the inhibitor. They also determined the structure of the synthase with the inhibitor bound to it by exposing crystals composed of the protein-inhibitor complex to high-energy X-rays from Argonne’s Advanced Photon Source (APS) and recording how the electrons in the molecule scattered the light.

“The collaboration made it possible for us to determine the allosteric nature of the inhibitor and the nuances of how the inhibition worked,” said Deborah Hung, a professor at the Harvard Medical School and co-director of the Infectious Disease and Microbiome Program at the Broad Institute.

The structural analysis determined that there are two key parts that make up tryptophan synthase. When one of the regions signals to the other that it is ready, the other region makes a chemical substance called indole. The indole then travels through a channel to the other region of the tryptophan synthase where it combines with another substance to create the final product, tryptophan.

A slight instability in the channel would allow the indole to travel to the other side, but the inhibitor is shaped so that it stabilizes the channel, preventing the indole from moving. Since the indole can’t reach the far side of the protein, the inhibitor blocks the production of tryptophan.

“It actually works in quite a sneaky way,” said Andrzej Joachimiak, Director of Argonne’s Structural Biology Center and the Midwest Center for Structural Genomics. “The inhibitor both forces the tryptophan synthase to produce the indole and prevents it from carrying out its ultimate duty.”

Although researchers are optimistic about this inhibitor’s potential as an antibiotic, it is still far from being an available pharmaceutical. They have studied how human tissue would react to the molecule and have confirmed that it is non-toxic, but experiments in mice have shown that the inhibitor would be metabolized by the body too quickly. Research is now being done to refine the structure of the inhibitor so it persists in the bloodstream for an extended period of time.

The research was funded by the National Institutes of Health, the Broad Institute Tuberculosis Donor Group, the Pershing Square Foundation and the Bill and Melinda Gates Foundation. Work done at the APS, a DOE Office of Science User Facility, was supported by DOE’s Office of Science.

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The U.S. Department of Energy's Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time. For more information, visit the Office of Science website.

Image of the protein tryptophan synthase created using diffraction data from Argonne's Advanced Photon Source. The inhibitor binds between the reaction sites in the protein, represented here by the orange and blue pockets. (Image by Argonne National Laboratory.)

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